title: The thermostability of two kinds of recombinant ∆6-fatty acid desaturase with different N-terminal sequence lengths in low temperature.
abstract: Two recombinant Rhizopus stolonifer ∆6-fatty acid desaturase enzymes with different-length N-termini were cloned and expressed in Saccharomyces cerevisiae strain INVScl: LRsD6D begins with the sequence of the N-terminal of the R. stolonifer ∆6-fatty acid desaturase native, encoding a deduced polypeptide of 459 amino acids (M-S-T-L-D-R-Q-S-I-F-T-I-K-E-L-E-S-I-S-Q-R-I-H-D-G-D-E-E-A-M-K-F), whereas SRsD6D begins with the amino acid sequence of the predicted ORF, encoding a deduced polypeptide of 430 amino acids (M-K-F) and LRsD6D is longer than SRsD6D by 29 amino acids (M-S-T-L-D-R-Q-S-I-F-T-I-K-E-L-E-S-I-S-Q-R-I-H-D-G-D-E-E-A). Bioinformatic analysis characterized the two recombinant ∆6-fatty acid desaturase enzymes with different-length N-termini, including three conserved histidine-rich motifs, hydropathy profile, and a cytochrome b5-like domain in the N-terminus. When the coding sequence was expressed in S. cerevisiae strain INVScl, the coding produced ∆6-fatty acid desaturase activity exhibited by RsD6D, leading to a novel peak corresponding to γ-linolenic acid methyl ester standards, which was detected with the same retention time. The residual activity of LRsD6D was 74 % at 15 °C for 4 h and that of SRsD6D was 43 %. Purified recombinant LRsD6D was more stable than SRsD6D, indicating that the N-terminal extension, containing mostly hydrophobic residues, affected the overall stability of recombinant LRsD6D.
authors.affiliation: Department of Microbiology, Chongqing University of Medical Sciences, Chongqing, 400016, People's Republic of China. yuxufei@126.com
authors.forename: He
authors.initials: H
authors.lastname: Lu
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D050602:Linoleoyl-CoA Desaturase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012233:Rhizopus / Q000201:enzymology / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D050602:Linoleoyl-CoA Desaturase